The present invention relates to the combined enrichment, processing and embedding of cytological specimens according to histological principles.
Most neoplastic and many other diseases are diagnosed by microscopic examination of cytological cell or of histological tissue specimens. New methods for obtaining cytological specimens from tissues, e.g., by aspiration using fine needles and by brushing material off mucosal membranes, have increased the demand for cytological methods that permit diagnostic accuracy comparable to that of histological specimens. Cytological and histological preparations should be thin enough to let pass the incident light of the microscope after staining and they should permit the use of all histochemical and immunological staining methods that are essential for accurate diagnoses.
Cytological specimens can be smeared onto slides and this originally haematological technique is used also for secretions and fine needle aspiration biopsies. Cells in fluids can be enriched onto filters (e.g. Millipore filters, Millipore Corporation, Bedford, Mass.) that permit the fluid proper to pass; in this technique the number of applicable staining methods is limited because the filters absorb many dyes. Cells can be centrifuged onto glass slides. Disadvantages common to these techniques are that specimens may contain cell clusters so large that they cannot be analysed since enough light does not pass them after staining and, furthermore, clusters of cells cannot easily be divided for staining by special techniques.
The above draw-backs do not apply to histological techniques and some prefer to concentrate and collect valuable cytological specimens to bodies that can be processed and embedded histologically. Fluid gelatin, agar, gels and plasma have been used as "glues" that can be solidified to form bodies that can be processed histologically. Cell clusters have been collected into funnels prepared from filter paper or plastic (Nordgren Hans, not published) nets, or onto polycarbonate filters (Nordgren et al. APMIS 97:136-42, 1989) that can be processed histologically. Fixed, repeatedly centrifuged and acetone-dehydrated specimens have been paraffinized after evaporation of the acetone, concentration of the specimen being obtained by centrifugation in the melted paraffin, but the method is not suitable to mucinous specimens (Krogerus & Anderssson. Acta Cytol 32: 585-7, 1988) and seems laborious.
Histological tissue specimens, after fixation, are transferred through a series of processing fluids that dehydrate and clear the tissues before they are impregnated by melted paraffin that upon cooling hardens to a block that is cut by a microtome, the thin sections after staining being examined microscopically. The tissue pieces are generally transferred to small perforated and labelled tissue casettes, which during processing are kept in large perforated baskets. After paraffination, the tissue pieces are transferred manually to metal molds for embedding, the molds being covered by the inverted labelled casette bottoms that after hardening of the paraffin remain attached to the paraffin block, the casette being clamped to the microtome jaws for cutting.
The latter laborious step of embedding has been considerably simplified by Pickett et al (U.S. Pat. No. 3,982,862). These authors transfer the fixed tissue piece directly into an open base pan, serving both as part of a tissue "casette" during paraffination and, after completed paraffination, as an embedding mold. For processing, the base pan is closed by a perforated and labelled top member that, after processing in a manner similar to the casette bottoms above, remains attached to the paraffin block and fits the jaws of a microtome. Adequate paraffination is achieved when, during processing, the top member closing the base pan is positioned vertically. However, the method by Pickett et al. is as unsuitable for cytological specimens as are conventional histological techniques.
In conventional automatic tissue processor types the baskets holding the casettes are lifted into the air when transferred from one processing fluid to the next one. In some modern processor types the baskets are kept in one single closed compartment in which the fluids are exchanged, periods of low pressure being applied to speed up paraffination.
The technical disadvantages of cytological techniques were mentioned above. From a cytological point of view the main disadvantages of histological techniques are that nucleated cells (diameter range some 10-20 .mu.m) and small cell clusters easily escape through histological casette perforations (&gt;1000 .mu.m) and that specimens are not adequately enriched unless they are "glued" into pieces corresponding in size to histological tissue ones.